For the breast cancer model, 7-week-old female Swiss nude mice (Charles River Laboratories) were engrafted subcutaneously with 5 x10⁶ MCF-7 cells 1 day after subcutaneous implantation of 0.72 mg 17-estradiol 60-day releasing pellets (Innovative Research of America) and treated with W0101 or isotype control ADC at 3 mg/kg every 4 days for 4 cycles.

Tumor and normal cells were plated in 96-well flat bottomed microplates (100 L/well) in cell culture medium and incubated overnight at 37°C in 5% CO₂. The next day, increasing concentrations of W0101 or isotype control ADC (010 ug/mL) were added into 3 replicate wells containing cells (10 L/well). Plates were incubated for 6 days at 37°C in 5% CO₂. Cell viability was determined by measuring ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was read using a Multimode Microplate Reader (Mithras LB940, Berthold Technologies). The percentage cell viability was calculated for each concentration considering 0 ug/mL ADC as 100% viability. The IC50 was calculated using Prism software. Three independent experiments were performed.

Tumor and normal cells were plated in 96-well flat bottomed microplates (100 L/well) in cell culture medium and incubated overnight at 37°C in 5% CO₂. The next day, increasing concentrations of W0101 or isotype control ADC (010 ug/mL) were added into 3 replicate wells containing cells (10 L/well). Plates were incubated for 6 days at 37°C in 5% CO₂. Cell viability was determined by measuring ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was read using a Multimode Microplate Reader (Mithras LB940, Berthold Technologies). The percentage cell viability was calculated for each concentration considering 0 ug/mL ADC as 100% viability. The IC50 was calculated using Prism software. Three independent experiments were performed.

Tumor and normal cells were plated in 96-well flat bottomed microplates (100 L/well) in cell culture medium and incubated overnight at 37°C in 5% CO₂. The next day, increasing concentrations of W0101 or isotype control ADC (010 ug/mL) were added into 3 replicate wells containing cells (10 L/well). Plates were incubated for 6 days at 37°C in 5% CO₂. Cell viability was determined by measuring ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was read using a Multimode Microplate Reader (Mithras LB940, Berthold Technologies). The percentage cell viability was calculated for each concentration considering 0 ug/mL ADC as 100% viability. The IC50 was calculated using Prism software. Three independent experiments were performed.

Tumor and normal cells were plated in 96-well flat bottomed microplates (100 L/well) in cell culture medium and incubated overnight at 37°C in 5% CO₂. The next day, increasing concentrations of W0101 or isotype control ADC (010 ug/mL) were added into 3 replicate wells containing cells (10 L/well). Plates were incubated for 6 days at 37°C in 5% CO₂. Cell viability was determined by measuring ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was read using a Multimode Microplate Reader (Mithras LB940, Berthold Technologies). The percentage cell viability was calculated for each concentration considering 0 ug/mL ADC as 100% viability. The IC50 was calculated using Prism software. Three independent experiments were performed.